Stabilizing formulation for NGF

ABSTRACT

Formulations are provided comprising NGF and acetate-containing buffer from pH 5 to 6 that provide enhanced stability of NGF for use in promoting nerve cell growth, repair, survival, differentiation, maturation or function.

[0001] This application is a continuation of U.S. patent applicationSer. No. 08/746,073, filed on Nov. 6, 1996, now U.S. Pat. No. 6,090,781,which claims priority to U.S. Provisional Patent Application No.60/046,874, having an effective filing date of Nov. 7, 1995, as properlyand timely obtained by the petition dated Nov. 5, 1996, under 37 CFR§1.53(b)(2)(ii) from U.S. patent application Ser. No. 08/554,685, filedNov. 7, 1995, the contents of which are incorporated herein byreference.

BACKGROUND

[0002] 1. Field of the Invention

[0003] This invention relates to formulations of nerve growth factor(“NGF”) and their use to induce nerve cell growth, differentiation,survival, repair, maturation, or function in vivo or ex vivo. Moreparticularly, this invention relates to such pharmaceutical compositionshaving increased stability and solubility characteristics for the NGFcomponent, particularly human recombinant NGF (“rhNGF”), and thosemaking possible the ability to create stable forms thereof for safe,effective therapeutic administration to human subjects.

[0004] 2. Description of Related Disclosures

[0005] Nerve growth factor (NGF) is a neurotrophic factor required forthe growth and survival of sympathetic and sensory neurons duringdevelopment and in mature animals (1). Clinical indications forrecombinant human NGF include peripheral sensory neuropathy andAlzheimer's disease. For example, the systemic administration of NGF hasbeen shown to reduce the sensory neuropathy induced by administration ofcisplatin and taxol to mice (2,3). In recent clinical trials, NGF hasbeen administered to humans to improve sensory function in diabeticneuropathies (4).

[0006] NGF is currently being developed as a liquid parenteralformulation. The protein stability is complicated beyond the usualchemical and physical degradation pathways due to the dimeric structureof NGF. Protein stability can be further complicated when recombinantprotein is a mixture of C-terminally clipped NGF variants. The crystalstructure of murine NGF shows 3 antiparallel pairs of b-strands forminga flat surface through which the monomers dimerize (5); the dimerdissociation constant is ≦10⁻¹³ M (6, 7). The rearrangement of monomerswithin dimers, towards an equilibrium dimer distribution, complicatesquantification of NGF dimer degradation.

[0007] There exists a need for formulations containing NGF that lead toNGF stability while being safe and effective for therapeuticadministration to mammals, particularly human subjects.

SUMMARY

[0008] The present invention is based on the finding of formulationconditions and methods for stability of NGF in a liquid formulation. Itis an object of the present invention to provide a suitable formulationof NGF with enhanced stability of NGF to provide effective induction ofnerve cell growth, survival, differentiation, maturation, repair, orfunction, preferably in vivo or ex vivo. In various embodiments theformulations can have enhanced stability to agitation, freezing,thawing, light, or storage. It is another object of the invention toprovide a stable NGF formulation for use in treating a mammal,preferably human, in need of NGF treatment so as to provide atherapeutically effective amount of NGF. It is further object to providean NGF formulation with enhanced consistency for improved application tothe neuron or mammal. These and other objects will become apparent tothose skilled in the art.

[0009] The above objects are achieved by providing an NGF formulationcomprising an effective amount of NGF in a pharmaceutically acceptableacetate buffer, preferably sodium acetate. In a specific embodiment thisformulation contains about 0.1 to 2.0 mg/ml NGF in an acetate bufferfrom 5 to 50 mM, from pH 5 to 6. The formulation can optionally containa pharmaceutically acceptable diluent, a pharmaceutically acceptablesalt, preferably sodium chloride, or a preservative, preferably benzylalcohol.

[0010] In another embodiment the invention provides a method ofproducing an NGF formulation produced by the steps including formulatingNGF and acetate, and optionally sodium chloride, and further optionallya preservative.

[0011] In another embodiment a method is presented by which NGF dimerdegradation is quantitated independent of dimer exchange.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 depicts the dependence of NGF aggregate formation at 37° C.on formulation buffer and pH, quantitated by size-exclusionchromatography, (⋄) succinate pH 4.2; (Δ) succinate pH 5.0; (□)succinate pH 5.8; (X) succinate pH 5.0 with 0.05% Tween 20; (▴) acetatepH 5.0; and (▪) acetate pH 5.8.

[0013]FIG. 2 depicts representative RP-HPLC chromatograms for NGF insuccinate buffer at pH 5.0 (a) −70° C. control and (b) after 38 days ofincubation at 37 degrees C.

[0014]FIG. 3 depicts semilogarithmic plot of the percent NGF monomerremaining after incubation at 37° C. for various lengths of time asquantitated by RP-HPLC, (⋄) succinate pH 4.2; (Δ) succinate pH 5.0; (□)succinate pH 5.8; (X) succinate pH 5.0 with 0.05% Tween 20; (▴) acetatepH 5.0; and (▪)acetate pH 5.8. Curves are first order fits to the data.

[0015]FIG. 4 depicts representative IEC chromatograms for NGF in acetatebuffer at pH 5.0 after 38 days of incubation at (solid line) −70° C. and(dashed line) 37° C. Each dimer appears as a triplet in the chromatogramdue to N-terminal Ser to Gly (S1G) conversion (13). The earliest peak inthe triplet is the parent dimer, followed by a dimer with a single Serto Gly conversion, and finally a dimer with a Ser to Gly conversion inboth chains.

[0016]FIG. 5 depicts time dependence of the loss of NGF 118/118 and117/120 dimers, by IEC, on incubation at 37° C., (Δ) succinate pH 5.0;(□) succinate pH 5.8; (X) succinate pH 5.0 with 0.05% Tween 20; (▴)acetate pH 5.0; and (▪) acetate pH 5.8.

[0017]FIG. 6 depicts RP-HPLC chromatograms showing the stability of NGFafter 1.6 years at

[0018] (dashed line) 5° C. and (solid line) −70° C. The majordegradation product at 5° C. is Asn93 to iso-Asp93 conversion.

[0019]FIGS. 7A and 7B depict comparisons of NGF (solid line) −70° C.control and (dashed line) 5° C. IEC chromatograms after 1.6 years ofincubation in acetate buffer at pH 5.0, (FIG. 7A) no acid treatment, and(FIG. 7B) acid treatment of samples prior to analysis.

[0020]FIG. 8 depicts RP-HPLC chromatograms of 0.1 mg/ml rhNGF in 10 mMacetate at pH 5.5 and 142 mM NaCl stored at 5° C. (solid line), 25° C.(dashed line), and 40° C. (dotted line) for 3 months. Peak (a) containsdi-oxidized rhNGF; peak (b) contains deamidated rhNGF; peak (c) containsmono-oxidized rhNGF; peak (d) contains Iso-aspartate; peak (e) contains120 rhNGF; peak (f) contains 118 rhNGF; peak (g) contains N-terminallyclipped rhNGF; peak (h) contains misfolded rhNGF; and peak (i) containsprotein eluted at gradient ramp.

[0021]FIG. 9 depicts determination of rhNGF monomers (118 and 120)remaining in rhNGF formulations after 12 months at 5 degrees C. byreversed-phase HPLC. Formulation A (-⊖-) contains 2 mg/ml rhNGF (142 mMNaCl, 10 mM acetate, pH 5.5); formulation B (-□-) contains 0.1 mg/mLrhNGF (136 mM NaCl, 20 mM acetate, pH 5.5); formulation C (--⋄--)contains formulation B plus 0.9% BA; formulation D (--x--) containsformulation B plus 0.25% phenol; formulation E (---+---) contains 0.1mg/mL rhNGF (136 mM NaCl, 20 mM acetate, 0.01% F68, pH 5.5); formulationF (-Δ--) contains formulation E plus 0.9% BA; and formulation G (--▪--)contains formulation E plus 0.25% phenol.

[0022]FIG. 10 depicts determination of rhNGF monomers (118 and 120)remaining in rhNGF formulations after 9 months at 25 degrees C. byreversed-phase —HPLC. Formulation A (-⊖-) contains 2 mg/ml (10 mMacetate, pH 5.5); formulation. B (-□-) contains 0.1 mg/ml (20 mMacetate, pH 5.5); formulation C (--⋄--) contains formulation B plus 0.9%BA; formulation D (--x--) contains formulation B plus 0.25% phenol;formulation E (--+--) contains 0.1 mg/mL (20 mM acetate, 0.01% F68, pH5.5); formulation F (-Δ--) contains formulation E plus 0.9% BA; andformulation G (----) contains formulation E plus 0.25% phenol.

[0023]FIG. 11 depicts effect of preservative on Iso-aspartate formationof rhNGF in liquid multi-dose formulations stored at 5 degrees C. for 12months as determined by RP-HPLC. Formulation A (-⊖-) contains 2 mg/mL(10 mM acetate, pH 5.5); formulation B (-□-) contains 0.1 mg/mL (20 mMacetate, pH 5.5); formulation C (--⋄--) contains formulation B plus 0.9%BA; formulation D (--x--) contains formulation B plus 0.25% phenol;formulation E (--+--) contains 0.1 mg/mL (20 mM acetate, 0.01% F68, pH5.5); formulation F (-Δ--) contains formulation E plus 0.9% BA; andformulation G (--▪--) contains formulation E plus 0.25% phenol.

[0024]FIG. 12 depicts effect of preservative on Iso-aspartate formationof rhNGF in liquid multi-dose formulations stored at 25 degrees C. for 9months as determined by RP-HPLC. Formulation A (-⊖-) contains 2 mg/mL(10 mM acetate, pH 5.5); formulation B (-□-) contains 0.1 mg/mL (20 mMacetate, pH 5.5); formulation C (--⋄--) contains formulation B plus 0.9%BA; formulation D (--x--) contains formulation B plus 0.25% phenol;formulation E (--+--) contains 0.1 mg/mL (20 mM acetate, 0.01% F68, pH5.5); formulation F (-Δ--) contains formulation E plus 0.9% BA; andformulation G (--▪--) contains formulation E plus 0.25% phenol.

[0025]FIG. 13 depicts cation exchange HPLC chromatograms of 0.1 mg/mlrhNGF in 10 mM acetate at pH 5.5 and 142 mM NaCl stored at 5 degrees C.(solid line), 25 degrees C. (dashed line), and 40 degrees C. (dottedline) for 3 months. Peak (a) contains mono and di-oxidized 118/118 andoxidized N-terminally clipped rhNGF; peak (b) contains 118/118 rhNGFhomodimer; and peak (c) contains Ser-Gly 118/118 rhNGF (1-chain).

[0026]FIG. 14 depicts determination of rhNGF dimer (118/118) remainingin rhNGF formulations after 12 months at 5 degrees C. by cation exchangeHPLC. Formulation A (-⊖-) contains 2 mg/mL (10 mM acetate, pH 5.5);formulation B (-□-) contains 0.1 mg/mL (20 mM acetate, pH 5.5);formulation C (--⋄--) contains formulation B plus 0.9% BA; formulation D(--x--) contains formulation B plus 0.25% phenol; formulation E (--+--)contains 0.1 mg/mL (20 mM acetate, 0.01% F68, pH 5.5); formulation F(-Δ--) contains formulation E plus 0;9% BA; and formulation G (----)contains formulation E plus 0.25% phenol.

[0027]FIG. 15 depicts determination of rhNGF dimer (118/118) remainingin rhNGF formulations after 9 months at 25 degrees C. by cation exchangeHPLC. Formulation A (-⊖-) contains 2 mg/mL (10 mM acetate, pH 5.5);formulation B (-□-) contains 0.1 mg/mL (20 mM acetate, pH 5.5);formulation C (--⋄--) contains formulation B plus 0.9% BA; formulation D(--x--) contains formulation B plus 0.25% phenol; formulation E (--+--)contains 0.1 mg/mL (20 mM acetate, 0.01% F68, pH 5.5); formulation F(-Δ--) contains formulation E plus 0.9% BA; and formulation G (----)contains formulation E plus 0.25% phenol.

[0028]FIG. 16 depicts near UV CD spectrum of rhNGF in 10 mM acetate, 136mM NaCl, pH 5.5.

[0029]FIG. 17 depicts a comparison of near-UV CD spectra of rhNGF in thepresence (solid line) and absence (dotted line) of 0.9% benzyl alcoholin 20 mM acetate at pH 5.5 and 136 mM NaCl after 24 hours at 25 degreesC.

DETAILED DESCRIPTION

[0030] The present invention is based on the discovery that NGFformulated in pharmaceutically acceptable acetate buffer from pH 5 to pH6 as a pharmaceutical composition has markedly increased stability inthese compositions. Acetate concentrations can range from 0.1 to 200 mM,more preferably from 1 to 50 mM, and even more 5 to 30 mM, and mostpreferably from 10 to 20 mM. One preferred embodiment has 20 mM acetateand another has 10 mM acetate in the administered solution. A preferredacetate salt for enhancing stability and buffering capacity is sodiumacetate. However other physiologically acceptable acetate salts can beused, for example potassium acetate. Suitable pH ranges for thepreparation of the compositions herein are from 5 to 6, preferably 5.4to 5.9, more preferably 5.5 to 5.8. A preferred pH is 5.5 which enhancesstability and buffering capacity. Another preferred embodiment is pH5.8, A “pharmaceutically effective amount” of NGF refers to that amountwhich provides therapeutic effect in various administration regimens.The compositions herein are prepared containing amounts of NGF from 0.07to 20 mg/ml, preferably 0.08 to 15 mg/ml, more preferably 0.09 to 10mg/ml, and most preferably 0.1 to 2 mg/ml. In a preferred embodiment theNGF concentration is 0.1 mg/ml. In another preferred embodiment the NGFconcentration is 2.0 mg/ml. For use of these compositions inadministration to human patients suffering from peripheral neuropathies,for example, these compositions may contain from about 0.1 mg/ml toabout 2 mg/ml NGF, corresponding to the currently contemplated dosagerate for such treatment. NGF is well-tolerated and higher doses can beadministered if necessary as determined by the physician.

[0031] Optionally, but preferably, the formulation contains apharmaceutically acceptable salt, preferably sodium chloride, andpreferably at about physiological concentrations. Low concentrations arepreferred, e.g., less than about 0.3 M to about 0.05 M, preferably from0.16 to 0.20 M NaCl, more preferably 0.13 to 0.15 M. In a preferredembodiment the sodium chloride concentration is 136 mM. In anotherpreferred embodiment the concentration is. 142 mM.

[0032] Optionally, the formulations of the invention can contain apharmaceutically acceptable preservative. In some embodiments thepreservative concentration ranges from 0.1 to 2.0%, typically v/v.Suitable preservatives include those known in the pharmaceutical arts.Benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben arepreferred preservatives. Benzyl alcohol is a particularly preferredpreservative that results in enhanced NGF stability. A particularlypreferred benzyl alcohol concentration is 0.7 to 1.2%, more preferably0.8 to 1.0%, with a particularly preferred concentration of 0.9%.

[0033] Optionally, the formulations of the invention can include apharmaceutically acceptable surfactant. Preferred surfactants arenon-ionic detergents. Preferred surfactants include Tween 20 andpluronic acid (F68). F68 is particularly preferred for enhancing NGFstability. Suitable surfactant concentrations are 0.005 to 0.02%. Apreferred concentration for surfactant is 0.01%. Surfactants are used tominimize particulate formation.

[0034] In a particularly preferred embodiment the composition containsan NGF concentration of 0.1 mg/ml, a sodium acetate concentration of 20mM, pH 5.5, a sodium chloride concentration of 136 mM, and benzylalcohol concentration at 0.9% (v/v). In another embodiment the NGFconcentration is 2.0 mg/ml, the sodium acetate concentration is 10 mM,pH 5.5, and the sodium chloride concentration is 142 mM.

[0035] In another embodiment of the invention is provided a kit for NGFadministration, which includes a vial or receptacle containing apharmaceutical composition of the invention comprising apharmaceutically effective amount of nerve growth factor and apharmaceutically acceptable acetate-containing buffer. A preferred vialvolume is one suitable for multi-dose use—allowing repeated withdrawalof sample. The increased stability attained with the formulations of theinvention allow multi-dose liquid formulation. Typically a multi-dosevial will provide sufficient formulation to supply sufficient dosage forone patient for one month, preferably one week. For example, thecomposition volume generally ranges from 0.3 to 10.0 ml and morepreferably from 1.6 to 2.0 ml, depending on dose concentration,frequency and ease of use. For example, a volume of 1.8 ml is convenientwhen either 0.3 ug/kg or 0.1 ug/kg are used, allowing 7 or 24 doses,respectively. When a light sensitive component, such as benzyl alcoholis present, the vial is protected from intense light. Generally it issufficient to store the vial in a darkened refrigerator or within anopaque box. However, the vial walls can comprise light transmissionreducing materials. For example, translucent amber or brown vials or anopaque vail can be used. In preferred embodiments the vial containsmulti-dose formulation. For a vial configuration, a selected multi-doseliquid formulation can be filled in 3 cc Type I glass vial with 1.8 mLfill volume. Selection of stopper will be based on compatibility ofdifferent types of stopper with the selected formulation.

[0036] Compositions of the invention are typically stored at 2 to 8degrees C. The formulations are stable to numerous freeze thaw cycles asshown herein.

[0037] In another embodiment the formulation is prepared with the aboveacetate concentrations.

[0038] A preferred means of preparing a formulation is to dialyze a bulkNGF solution into the final formulation buffer. Final NGF concentrationsare achieved by appropriate adjustment of the formulation withformulation buffer absent NGF. Also provided are methods for thepreparation of the composition of claim 1 comprising the steps ofcompounding said NGF and acetate-containing buffer. Also provided aremethods of increasing the stability of NGF in a pharmaceuticalcomposition containing NGF as active principle, comprising incorporatingacetate in said composition, wherein said acetate is present in anamount and pH effective to increase the stability of the NGF.

[0039] The compositions hereof including lyophilized forms, are preparedin general by compounding the components using generally availablepharmaceutical compounding techniques, known per se. Likewise, standardlyophilization procedures and equipment well-known in the art areemployed. A particular method for preparing a pharmaceutical compositionof NGF hereof comprises employing purified (according to any standardprotein purification scheme) NGF, preferably rhNGF, in any one ofseveral known buffer exchange methods, such as gel filtration ordialysis.

[0040] Nerve growth factor (“NGF”) is a 120 amino acid polypeptidehomodimeric protein that has prominent effects on developing sensory andsympathetic neurons of the peripheral nervous system. NGF acts viaspecific cell surface receptors on responsive neurons to supportneuronal survival, promote neurite outgrowth, and enhance neurochemicaldifferentiation. NGF actions are accompanied by alterations in neuronalmembranes, in the state of phosphorylation of neuronal proteins, and inthe abundance of certain mRNAs and proteins likely to play a role inneuronal differentiation and function. (Connolly et al., J. Cell. Biol.90:176-180 [1981]; Skaper and Varon, Brain Res. 197:379-389 [1980]; Yu,et al., J. Biol. Chem. 255:10481-10492 [1980]; Haleqoua and Patrick,Cell 22:571-581 [1980]; Tiercy and Shooter, J. Cell. Biol. 103:2367-2378[1986]).

[0041] Forebrain cholinergic neurons also respond to NGF and may requireNGF for trophic support. (Hefti, J. Neurosci., 6: 2155 [1986]). Indeed,the distribution and ontogenesis of NGF and its receptor in the centralnervous system (CNS) suggest that NGF acts as a target-derivedneurotrophic factor for basal forebrain cholinergic neurons (Korsching,TINS, pp 570-573 [November/December 1986]).

[0042] Little is known about the NGF amino acid residues necessary forthe interaction with the trkA-tyrosine kinase receptor. Significantlosses of biological activity and receptor binding were observed withpurified homodimers of human and mouse NGF, representing homogenoustruncated forms modified at the amino and carboxy termini. The 109 aminoacid species (10-118)hNGF, resulting from the loss of the first 9residues of the N-terminus and the last two residues from the C-terminusof purified recombinant human NGF, is 300-fold less efficient indisplacing mouse [¹²⁵I]NGF from the human trkA receptor compared to(1-118)hNGF. It is 50- to 100-fold less active in dorsal root ganglionand sympathetic ganglion survival compared to (1-118)hNGF. The(1-118)hNGF has considerably lower trkA tyrosine kinaseautophosphorylation activity. A preferred form is the 118 amino acidhuman NGF, which is more preferable as a homodimer.

[0043] The formulations of the invention include the pantropicneurotrophin pantropic NGF. Pantropic NGF is a pantropic neurotrophinwhich has an amino acid sequence homologous to the amino acid sequenceof NGF, with domains which confer other neurotrophin specificities. Inthe preferred embodiment, the domains are substituted for NGF residues;that is, some number of amino acids are deleted from the NGF sequence,and an identical or similar number of amino acids are substituted,conferring an additional specificity. For example, a pantropic NGF ismade with a D16A substitution, which confers BDNF specificity.Optionally, substitutions in the pre-variable region 1 (V18E+V20L+G23T)and in variable region 4 (Y79Q+T81K⁺ H84Q+F86Y+K88R) are included.Alternatively, the substitutions in the pre-variable region 1 can bemade with only single amino acid substitutions in variable region 4; forexample, V18E+V20L+G23T and one of Y79Q, T81K, H84Q, F86Y, or K88R maybe made.

[0044] The chemical and physical stability of recombinant human nervegrowth factor (NGF) in aqueous solution was investigated between 5 and37° C., in the pH range 4.2 to 5.8. NGF chemical stability increasedwith increasing pH. In succinate buffer at pH 5.8, NGF physicalstability decreased due to protein aggregation. Based on both the 5° C.stability data and accelerated degradation studies at 37° C., theoptimal formulation was found to be acetate buffer at pH 5.8.Reversed-phase HPLC was the primary stability indicating method, showingconversion of Asn-93 to iso-Asp to be the primary degradation pathway at5° C. Quantitation of NGF degradation by cation exchange chromatographywas complicated by the rearrangement of the NGF monomer variants intovarious mixed dimers over time (dimer exchange). Treatment of samplesand controls with dilute acid rapidly equilibrated the monomerdistribution in the dimers, allowing NGF degradation to be quantitatedin the absence of dimer exchange.

[0045] Benzyl alcohol and phenol were evaluated for their compatibilityand stability with rhNGF in two liquid formulations for multi-usepurposes. These two formulations consist of 0.1 mg/mL protein in 20 mMsodium acetate at pH 5.5 and 136 mM sodium chloride with and without0.01% pluronic acid (F68) as surfactant. The final concentrations ofbenzyl alcohol and phenol in each of these two formulations were 0.9 and0.25%, respectively. Based on the 12 month stability data, rhNGF is morestable with benzyl alcohol than phenol in these formulations. Benzylalcohol preserved rhNGF formulation with the presence of surfactant isas stable as the formulation with no surfactant added, indicating thatthe addition of F68 to rhNGF multi-dose formulation is not required forstability purpose. Therefore, a formulation consisting of 0.1 mg/mLprotein in 20 mM acetate, 136 mM NaCl, 0.9% benzyl alcohol*, pH 5.5 isrecommended for rhNGF used for multiple dosing in Phase III clinicaltrails. This rhNGF multi-dose formulation passed the USP and EPpreservative efficacy test after 6 months at 5 degrees C., and is asstable as the current liquid formulation at 2 mg/mL. However, theformulation should avoid exposure to intensive light due to the presenceof benzyl alcohol as preservative which is light sensitive.

[0046] In general, the compositions may contain other components inamounts preferably not detracting from the preparation of stable, liquidor lyophilizable forms and in amounts suitable for effective, safepharmaceutical administration.

[0047] In order that materials like NGF be provided to health carepersonnel and patients, these materials must be prepared aspharmaceutical compositions. Such compositions must be stable forappropriate periods of time, must be acceptable in their own right foradministration to humans, and must be readily manufacturable. An exampleof such a composition would be a solution designed for parenteraladministration. Although in many cases pharmaceutical solutionformulations are provided in liquid form, appropriate for immediate use,such parenteral formulations may also be provided in frozen or inlyophilized form. In the former case, the composition must be thawedprior to use. The latter form is often used to enhance the stability ofthe medicinal agent contained in the composition under a wider varietyof storage conditions, as it is recognized by those skilled in the artthat lyophilized preparations are generally more stable than theirliquid counterparts. Such lyophilized preparations are reconstitutedprior to use by the addition of suitable pharmaceutically acceptablediluent(s), such as sterile water for injection or sterile physiologicalsaline solution, and the like.

[0048] NGF formulations of the invention are believed to be useful inpromoting the development, maintenance, or regeneration of neurons invivo, including central (brain and spinal chord), peripheral(sympathetic, parasympathetic, sensory, and enteric neurons), andmotorneurons. Accordingly, NGF formulations of the invention areutilized in methods for the treatment of a variety of neurologicdiseases and disorders. In a preferred embodiment, the formulations ofthe present invention are administered to a patient to treat neuraldisorders. By “neural disorders” herein is meant disorders of thecentral and/or peripheral nervous system that are associated with neurondegeneration or damage. Specific examples of neural disorders include,but are not limited to, Alzheimer's disease, Parkinson's disease,Huntington's chorea, stroke, ALS, peripheral neuropathies, and otherconditions characterized by necrosis or loss of neurons, whethercentral, peripheral, or motorneurons, in addition to treating damagednerves due to trauma, burns, kidney disfunction, injury, and the toxiceffects of chemotherapeutics used to treat cancer and AIDS. For example,peripheral neuropathies associated with certain conditions, such asneuropathies associated with diabetes, AIDS, or chemotherapy may betreated using the formulations of the present invention. It also isuseful as a component of culture media for use in culturing nerve cellsin vitro or ex vivo.

[0049] In various embodiments of the invention, NGF formulations areadministered to patients in whom the nervous system has been damaged bytrauma, surgery, stroke, ischemia, infection, metabolic disease,nutritional deficiency, malignancy, or toxic agents, to promote thesurvival or growth of neurons, or in whatever conditions have been foundtreatable with NGF. For example, NGF formulation of the invention can beused to promote the survival or growth of motorneurons that are damagedby trauma or surgery. Also, NGF formulations of the invention can beused to treat motoneuron disorders, such as amyotrophic lateralsclerosis (Lou Gehrig's disease), Bell's palsy, and various conditionsinvolving spinal muscular atrophy, or paralysis. NGF formulations of theinvention can be used to treat human neurodegenerative disorders, suchas Alzheimer's disease, Parkinson's disease, epilepsy, multiplesclerosis, Huntington's chorea, Down's Syndrome, nerve deafness, andMeniere's disease. NGF formulations of the invention can be used ascognitive enhancer, to enhance learning particularly in dementias ortrauma. Alzheimer's disease, which has been identified by the NationalInstitutes of Aging as accounting for more than 50% of dementia in theelderly, is also the fourth or fifth leading cause of death in Americansover 65 years of age. Four million Americans, 40% of Americans over age85 (the fastest growing segment of the U.S. population), haveAlzheimer's disease. Twenty-five percent of all patients withParkinson's disease also suffer from Alzheimer's disease-like dementia.And in about 15% of patients with dementia, Alzheimer's disease andmulti-infarct dementia coexist. The third most common cause of dementia,after Alzheimer's disease and vascular dementia, is cognitive impairmentdue to organic brain disease related directly to alcoholism, whichoccurs in about 10% of alcoholics. However, the most consistentabnormality for Alzheimer's disease, as well as for vascular dementiaand cognitive impairment due to organic brain disease related toalcoholism, is the degeneration of the cholinergic system arising fromthe basal forebrain (BF) to both the codex and hippocampus (Bigl et al.in Brain Cholinergic Systems, M. Steriade and D. Biesold, eds., OxfordUniversity Press, Oxford, pp.364-386 (1990)). And there are a number ofother neurotransmitter systems affected by Alzheimer's disease (DaviesMed Res. Rev.3:221 (1983)). However, cognitive impairment, related forexample to degeneration of the cholinergic neurotransmitter system, isnot limited to individuals suffering from dementia. It has also beenseen in otherwise healthy aged adults and rats. Studies that compare thedegree of learning impairment with the degree of reduced corticalcerebral blood flow in aged rats show a good correlation (Berman et al.Neurobiol. Aging 9:691 (1988)). In chronic alcoholism the resultantorganic brain disease, like Alzheimer's disease and normal aging, isalso characterized by diffuse reductions in cortical cerebral blood flowin those brain regions where cholinergic neurons arise (basal forebrain)and to which they project (cerebral cortex) (Lofti et al., Cerebrovasc.and Brain Metab. Rev 1:2 (1989)). Such dementias can be treated byadministration of NGF formulations of the invention.

[0050] Further, NGF formulations of the invention are preferably used totreat neuropathy, and especially peripheral neuropathy. “Peripheralneuropathy” refers to a disorder affecting the peripheral nervoussystem, most often manifested as one or a combination of motor, sensory,sensorimotor, or autonomic neural dysfunction. The wide variety ofmorphologies exhibited by peripheral neuropathies can each be attributeduniquely to an equally wide number of causes. For example, peripheralneuropathies can be genetically acquired, can result from a systemicdisease, or can be induced by a toxic agent. Examples include but arenot limited to diabetic peripheral neuropathy, distal sensorimotorneuropathy, or autonomic neuropathies such as reduced motility of thegastrointestinal tract or atony of the urinary bladder. Examples ofneuropathies associated with systemic disease include post-poliosyndrome; examples of hereditary neuropathies includeCharcot-Marie-Tooth disease, Refsum's disease, Abetalipoproteinemia,Tangier disease, Krabbe's disease, Metachromatic leukodystrophy, Fabry'sdisease, and Dejerine-Sottas syndrome; and examples of neuropathiescaused by a toxic agent include those caused by treatment with achemotherapeutic agent such as vincristine, cisplatin, methotrexate, or3′-azido-3′-deoxythymidine.

[0051] A therapeutically effective dose of an NGF formulation isadministered to a patient. By “therapeutically effective dose” herein ismeant a dose that produces the effects for which it is administered. Theexact dose will depend on the disorder to be treated, and will beascertainable by one skilled in the art using known techniques. Ingeneral, the NGF formulations of the present invention are administeredat about 0.01 μg/kg to about 100 mg/kg per day. Preferably, from 0.1 to0.3 ug/kg. In addition, as is known in the art, adjustments for age aswell as the body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the disease may benecessary, and will be ascertainable with routine experimentation bythose skilled in the art. Typically, the clinician will administer NGFformulations of the invention until a dosage is reached that repairs,maintains, and, optimally, reestablishes neuron function. The progressof this therapy is easily monitored by conventional assays.

[0052] A “patient” for the purposes of the present invention includesboth humans and other mammals. Thus the methods are applicable to bothhuman therapy and veterinary applications.

[0053] Therapeutic formulations of NGF are prepared by mixing NGF havingthe desired degree of purity with optional physiologically acceptablecarriers, excipients or stabilizers (Remington's PharmaceuticalSciences). Acceptable carriers, excipients or stabilizers are nontoxicto recipients at the dosages and concentrations employed and will notsignificantly decrease NGF stability in the formulations as taughtherein. Such compounds include antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone, amino acids such as histidine, methionine,glycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or non-ionicsurfactants such as Tween, Pluronics or PEG.

[0054] NGF formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes. Ordinarily NGF formulations of the presentinvention will be stored in liquid form at 2 to 8 degrees C. Theformulations are suitable for frozen storage with repeated cycles ofthawing and freezing.

[0055] Therapeutic NGF compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

[0056] NGF optionally is combined with or administered in concert withother neurotrophic factors including NT-4/5, NT-3, and/or BDNF and isused with other conventional therapies for nerve disorders.

[0057] The administration of the formulations of the present inventioncan be done in a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, or intraocularly. The formulations can beadministered continuously by infusion into the fluid reservoirs of theCNS, although bolus injection is acceptable, using techniques well knownin the art, such as pumps or implantation. In some instances, forexample, in the treatment of wounds, the formulations may be directlyapplied as a solution or spray.

[0058] The following examples are offered by way of illustration and notby way of limitation. The disclosures of all citations in thespecification are expressly incorporated herein by reference.

EXAMPLES Example I

[0059] Materials

[0060] Recombinant human nerve growth factor (NGF) was produced inChinese hamster ovary cells and purified by reversed-phase (RP-HPLC) andion-exchange chromatography (IEC) as described previously (8). HPLCgrade acetonitrile, and TFA were used for RP-HPLC. All other chemicalswere USP grade. Sterile type I, clear glass, 2 cc vials were purchasedfrom Wheaton and used with siliconized, Teflon-coated, butyl rubberstoppers.

[0061] Methods

[0062] NGF was dialyzed into 10 mM sodium acetate, 142 mM sodiumchloride, at pH 5.0 and 5.8, and into 10 mM sodium succinate, 142 mMNaCl, at pH 4.2, 5.0, and 5.8, and adjusted to 10 mg/ml. Tween 20 wasalso added to a succinate pH 5.0 formulation to determine if surfactantwould reduce NGF aggregation (10 mM sodium succinate, 142 mM NaCl, 0.05%Tween 20).

[0063] Vials were aseptically filled with 0.3 ml of NGF formulation andstored at 5, 25, and 37° C. (25° C. data not reported here). Controlswere stored at −70° C. where no significant degradation has beenobserved. At each time point, 50 μl aliquots were removed fromindividual vials and stored at −70° C. until analysis.

[0064] HPLC Analysis. Cation exchange HPLC (IEC) was carried out on a HP1090 system using a Tosohas sulpho-propyl TSK-SP-5PW (7.5×75 mm) columnwith 10 m particles. Mobile phases were (A) 10 mM sodium phosphate, 5%(v/v) acetonitrile, pH 7.0 and (B) A+1.0 M ammonium chloride. NGF waseluted at 35° C. (0.5 ml/min) with a linear gradient of 20-40% B from 5to 60 minutes. The control and 1.6 year samples at 5° C. were alsoassayed after “acid-treatment” to bring the distribution of monomervariants in the dimers to equilibrium (8, 9). These samples wereadjusted to pH 3.5 with HCl and incubated at 37° C. for 2 hours (resultsat 2 and 4 hours were equivalent). A YMC C4, 5 μm (4.6×250 mm) columnwas used for reversed-phase HPLC (RP-HPLC) on a HP 1090 system at 25° C.NGF was eluted (0.5 m/min) using a linear gradient of 26-30% B in A(B=0.05% TFA in acetonitrile and A=0.05% TFA in water) run between 5 and40 minutes. Size exclusion HPLC (“SEC-HPLC”) was carried out using aPerkin Elmer Series 410 Bio LC Pump with a Perkin Elmer LC 90Spectrophotometric UV Detector and a Tosohas TSK 2000 SWXL, 5 μm(7.8×300 mm) column. This SEC column was run at 0.5 ml/min using a 0.2 Mpotassium phosphate, 0.45 M potassium chloride mobile phase, at pH 7.0.For SEC UV detection was at 280 nm; for RP-HPLC and IEC, at 214 nm. Forall assays 50 mg of NGF were injected.

[0065] SDS-PAGE. Samples were diluted into Novex tricine SDS samplebuffer and incubated for 1 hour at 50° C. Non-reduced SDS-PAGE was runon Novex tricine gels containing 10% acrylamide followed by CoomassieBlue staining. Molecular weights were estimated using Bio-Rad lowmolecular weight markers. Neurite Outgrowth Assay. The biologicalactivity of NGF was determined using the PC 12 assay developed by Greene(10) and modified as described by Schmelzer et al (8).

[0066] Hemolysis. All formulations were tested for hemolytic activity.The hemolysis procedure was that of Reed and Yalkowsky (11) except thatequal volumes of washed human red blood cells and formulation wereincubated at 37° C. for 30 minutes before analysis.

[0067] Results

[0068] Formulation development of NGF requires condition be found forwhich the protein shows ≧1.5 years of chemical and physical stability at2-8° C. We determined the approximate pH of maximal NGF stability byascertaining NGF stability in succinate buffer at pH 4.2, 5.0, and 5.8,and acetate buffer at pH 5.0 and 5.8. NGF stability decreases above pH6.0. The assays used to measure protein stability were IEC, SEC,RP-HPLC, SDS-PAGE, and the PC12 bioactivity assay. Formulationbiocompatibility was determined by hemolysis testing.

[0069] Stability of NGF at 37° C.

[0070] Aggregation of NGF. The dimer/monomer equilibrium constant formurine NGF is smaller than 10-13 M at pH 4-7 (6, 7, 9, 12). NGF,therefore, assayed primarily as a dimer in the neutral pH SEC assay. Asmall amount of aggregated NGF (tetramer based on molecular weightstandards) was observed in the control sample. This tetramer peak areaincreased with time at 37° C. A leading shoulder on this peak,indicating larger aggregates, was observed for all formulations after 38days at 37° C. The time dependencies of aggregate formation for thevarious formulations are shown in FIG. 1. The succinate pH 5.8formulation had the greatest aggregation rate. All other formulationshad similar rates of aggregate formation. The addition of the surfactantTween 20 offered no protection against aggregation in the pH 5.0succinate formulation. During preparation, the NGF pH 5.8 succinateformulation had to be filtered through a 47 mm diameter 0.22 mm filter,whereas all other formulation were filterable through a 25 mm diameterfilter. This is consistent with the high rate of aggregation observed at37° C. in succinate buffer at pH 5.8.

[0071] Aggregation was also monitored using non-reduced SDS-PAGE (gelsnot shown). In the −70° C. control samples 3 bands were observed:monomer at 13.5 kDa, a very faint dimer band at approximately 26 kDa,and a slightly more intense band at 31 kDa. The 26 and 31 kDa bandsbecame more intense on incubation at elevated temperatures. A smallamount of large molecular weight aggregate (>97 kDa) was observed in allformulations after 38 days at 37° C. The intensity of this band wasgreatest in the pH 5.8 succinate formulation, consistent with the poorfilterability and high aggregation rate observed by SEC for thisformulation. Tween 20 prevented the formation of this high molecularweight aggregate at pH. 5.0. With the exception of succinate at pH 5.8,these sizing methods do not differentiate between the quality of the NGFformulations.

[0072] NGF Monomer and Degradation Product Quantitation. The NGF used inthese studies consisted of a 1:9:1 ratio of the three monomericpolypeptides containing 120, 118, and 117 amino acids. The 118 aminoacid variant was produced by clipping of Ala120 and Arg119 from theC-terminus of the 120 parent; the 117 variant had an additional clip,Arg118 (8). At pH 5.0, the 117 variant has two fewer positive charges,and the 118 variant one fewer positive charge than the 120 parent. Thereis no significant difference in the bioactivity of the homodimers andheterodimers formed by the 117, 118, and 120 variants as measured by thePC12 and chick dorsal root ganglion assays (8). In the acidic, organic,RP mobile phase where NGF dissociates to monomer (8), the elution orderis 120 before 118, then 117. Typical RP-HPLC chromatograms for NGFstored in pH 5.0 succinate buffer, for 38 days, at −70° C. and 37° C.are shown in FIG. 2. At elevated temperature, peak area is lost from thepeaks defined as NGF (the sum of the 118 and 120 monomer peaks) with theiso-Asp, oxidized, and other NGF degradation peaks increasing in area.The 117 peak area was not included in the definition of NGF due tocoelution of degradation products with this peak at elevatedtemperatures. The time dependence of NGF degradation at 37° C., and theapparent first order rate constants for this degradation, are shown inFIG. 3 and Table 1, respectively. TABLE 1 Apparent First-Order RateConstants for NGF Degradation at 37° C. as Determined by RP-HPLC. BufferpH k (day-1) Succinate 4.2 2.2 × 10⁻² ± 1.0 × 10⁻³ 5.0 1.1 × 10⁻² ± 6.3× 10⁻⁴ (+Tween 20) 5.0 1.1 × 10⁻² ± 7.1 × 10⁻⁴ 5.8 5.7 × 10⁻³ ± 9.7 ×10⁻⁴ Acetate 5.0 7.9 × 10⁻³ ± 8.0 × 10⁻⁴ 5.8 4.0 × 10⁻³ ± 2.9 × 10⁻⁴

[0073] NGF stability decreased as the pH was lowered. In both theacetate and succinate pH 5.8 buffers NGF stability was greater than atpH 5.0. In succinate buffer at pH 4.2, the NGF degradation rate isfurther increased, with several hydrophobic degradation products beingobserved, possibly due to acid-induced cleavage at the Asp60-Pro61linkage. Tween 20 had no affect on NGF stability in succinate buffer atpH 5.0 (FIG. 3). The acetate formulation appears to be somewhat betterin maintaining NGF stability.

[0074] NGF Dimer Distribution. The three NGF monomers containing 117,118, and 120 amino acids may combine to form the 117/117, 118/118 and120/120 homodimers and the 117/118, 118/120, and 117/120 heterodimers.Association of these NGF variants has been shown to be random, with nomonomer appearing to prefer any other (8, 9). The dynamic dissociationand reassociation of monomers to form various dimers (dimer exchange) isaccelerated by low pH and increased temperature (9). For a randomassociation process at equilibrium, and an initial 117/118/120 ratio of1:9:1, the 118/118 homodimer will be the dominant dimer species withsmaller amounts of the 117/118 and 118/120 dimers being formed.

[0075] The 118/118 and 117/120 dimers have the same effective net chargein the chosen IEC mobile phase and therefore coelute on IEC during NGFpurification. This results in an initial non-equilibrium distribution ofthe monomer variants in NGF dimers in the NGF product. The 117/120 and118/118 dimers dissociate giving the 117 and 120 monomers which willreassociate most frequently with 118 monomer to form 117/118 and 118/120dimers. Due to the different charges on the monomers, the expectedelution order of these dimers on cation-exchange chromatography is:

[0076] 117/117<117/118<118/118=117/120<118/120<120/120.

[0077] The most populated dimers are distinguishable by IEC (8) as shownin FIG. 4.

[0078] Representative IEC chromatograms for NGF at pH 5.0 in succinatebuffer after 38 days at −70° C. and 37° C. are shown in FIG. 4. DuringNGF production, a fraction of the N-terminal serine residues areconverted to glycine with no affect on NGF activity (13). NGF isquantitated here as the sum of the 118/118 homodimer and the 118/118dimer with a Ser1 to Gly1 conversion in one of the two monomers (13)(and any coeluting 117/120 variants); the 117/118 and 118/120 peak areasare not included due to degradation products coeluting with these peaks.The rate of loss of NGF, as monitored by IEC at 37° C., is shown in FIG.5. The degradation kinetics for the 118 dimer are multiphasic. The lossin main peak area before 13 days is largely due to rearrangement of themonomer variants between the possible dimer types. The data after 13days more accurately describes NGF chemical degradation. NGF is moststable in the acetate formulations at pH 5.0 and 5.8, which have similarstability. NGF in succinate buffer at pH 5.8, and pH 5.0, with andwithout 0.05% Tween 20, all have similar stabilities. The hemolyticactivity of each of the NGF formulations was also tested. None of theformulations showed significant red blood cell hemolysis (<0.1%). Thebioactivity of NGF in each of the formulations was also determined,using the neurite extension PC12 assay. NGF was bioactive in all of theformulations after 38 days at 37° C. The large assay variability(approximately 50% error) did not allow quantitative bioactivitydifferences between these formulations to be determined.

[0079] A liquid formulation for NGF preferably has an adequateshelf-life at 5° C. The accelerated stability data at 37° C. showed NGFto be most stable in acetate buffer. Based on this data, NGF stabilityin the acetate pH 5.0 and 5.8 formulations was investigated for 1.6years at 5° C. RP-HPLC chromatograms at pH 5.0 for the 1.6 year −70° C.control and 5° C. samples are shown in FIG. 6. The major degradationproduct was Asp-93 conversion to iso-Asp; smaller amounts of Met-37 andMet-92 oxidation were observed. The apparent first order rate constantsfor NGF degradation, quantitated by RP-HPLC, are 1.4×10−4±1.7×10−5 d−1and 6.8×10−5±7.0×10−6 d−1 at pH 5.0 and 5.8, respectively. At 5° C., IECshows that NGF stability is approximately the same at pH 5.0 and 5.8,consistent with the 37° C. IEC data. Aggregation of the NGF dimers wasnot a significant degradation pathway at 5° C., only a 1% increase inaggregate was observed over 1.6 years of storage at 5° C.

[0080] The interpretation of the IEC data at both 5° C. and 37° C., iscomplicated by dimer exchange, the exchange rate being slower at thelower temperature. To improve IEC quantitation, the dimer distributionwas brought to equilibrium by incubation at pH 3.5 for 2 hours at 37° C.prior to IEC analysis (8,9,14). No new degradation products wereobserved after this treatment. The acetate pH 5.8 samples after 1.6years of incubation at 5° C. are compared with controls before and after“acid treatment” in FIG. 7. The loss of main peak area to the peripheralpeaks due to dimer exchange was eliminated by acid treatment, revealingthe true degradation of NGF. Quantitation after acid treatment showedthat 94 and 92% of the NGF main peaks remain after 1.6 years at 5° C. atpH 5.0 and pH 5.8, respectively, compared to 84 and 87% without acidtreatment. For comparison, RP-HPLC analysis showed 93 and 96% of the NGF118 and 120 monomers remaining at pH 5.0 and pH 5.8, respectively.

[0081] NGF chemical stability was shown to increase with pH, the pH ofmaximal stability being near pH 5.8. At a fixed pH, the RP-HPLC and IECdata at 5 and 37° C. were consistent in showing NGF chemical stabilityto be greater in acetate than succinate buffer. In addition, NGFaggregation was not a significant degradation pathway, except at pH 5.8in succinate buffer. A complicating factor in the determination of NGFstability is that dimer exchange contributes to the apparent degradationof NGF dimers as determined by IEC. A more accurate representation ofNGF chemical degradation can be obtained by pretreating the controls andsamples with acid at 37° C. to bring the dimer distribution toequilibrium. Taken together, these data show that the optimalformulation and storage conditions for NGF stability are acetate butterat pH 5.8 with storage at 5° C.

Example II

[0082] Results from Phase II clinical trials indicate that patients withperipheral neuropathy disease require three dosings per week of rhNGF ateither 0.3 or 0.1 μg/kg. This means that only 21 or 7 μg per dosing ofrhNGF is needed for an average patient of body weight 70 kg. Using thecurrent rhNGF liquid formulation (2 mg/mL in 10 mM sodium acetate, pH5.5, 142 mM NaCl) and vial configuration (0.7 mL per vial) would havewasted a lot of drug product. Therefore, a new rhNGF formulation at lowconcentration, preferably multi-dose configuration, is required toreduce the cost and wastage of the product. The purpose of this studywas to develop a stable multi-dose liquid formulation for rhNGF at 0.1mg/mL with 1.8 mL fill in 3 cc glass vial for use in Phase III clinicaltrails. With this new configuration, each vial will give 180 μg proteinand will provide at least 7 doses at the high dosing level (0.3 μg/kg)and 24 doses at the low dosing level (0.1 μg/mL).

[0083] In this study, the results on compatibility and stability ofpreservative containing 0.1 mg/ml rhNGF multi-dose liquid formulationsat pH 5.5 are presented. A comparison between the stability of the newmulti-dose liquid formulations at 0.1 mg/mL rhNGF and the current 2mg/mL rhNGF formulation was also studied. Results on agitation, freezingand thawing, and light compatibility studies of the lead multi-doseliquid formulations for 0.1 mg/mL rhNGF were also reported.

[0084] In this study, rhNGF concentrated bulk formulated at 11.6 mg/mLin 10 mM sodium acetate, 142 mM sodium chloride at pH 5.5 with 20 mLfilled in 100 cc glass vials was used. All chemical reagents andmaterials used in this Example are listed in Table 2. TABLE 2 List ofMaterials rhNGF concentrated bulk, 11.6 mg/mL, in 10 mM sodium acetate,142 mM sodium chloride, pH 5.5 Sodium acetate trihydrate, GenentechRelease Materials Code G20136, Lot #S0766 Glacial acetic acid, ReleaseMaterials Code G20027-01, Lot S0567 Sodium Chloride, Release MaterialsCode G20136, Lot S1152 Benzyl alcohol, Release Materials Code G20226,Lot C0200 m-cresol, Sigma, Lot 107F-3497 Methylparaben, Napp ChemicalInc., Lot LM 86-6285 Propylparaben, Napp Chemical Inc., LL86-6241Phenol, Release Materials Code G20136, Lot 620015, Lot B0901 Polysorbate20, Release Materials Code G20091, Lot A1408 Pluronic acid (F68),Release Materials Code GXXXX, Lot XXXX Sterile, pryogen-freenon-siliconized Type I clear glass 3 cc vials (Wheaton Tubing Products);prepared in Phase V per standard procedures Sterile 13 mm Purcoat rubberstoppers, Clinical manufacturing, Genentech, Inc. 13 mm aluminumflip-off cap, Clinical manufacturing, Genentech, Inc.

[0085] Methods

[0086] rhNGF Multi-dose Liquid Formulations Preparation. rhNGFconcentrated bulk was dialyzed into a formulation buffer consisting of20 mM sodium acetate, 136 mM sodium chloride at pH 5.5 byultrafiltration using Amicon Centriprep™ concentrator with molecularweight cutoff of 10,000 KD. This reformulated rhNGF bulk was thendiluted to 0.15 mg/mL using the same formulation buffer for dialysis.Preservatives and surfactants used for compatibility screening andformulation development studies were added to this diluted rhNGFsolution at their tested concentrations. Protein concentration for eachformulation was then adjusted to 0.1 mg/mL by UV analysis using theappropriate formulation buffer. A list of preservatives and theirconcentrations used for physical compatibility with rhNGF in liquidformulations are given in table 3. TABLE 3 List of PreservativeScreening Formulations for 0.1 mg/mL rhNGF Formulation buffer SurfactantPreservative  20 mM acetate, pH 5.5 none  0.9% benzyl alcohol 136 mMNaCl 0.25% phenol 0.45% phenol 0.25% m-cresol 0.18% methylparaben 0.02%propylparaben  20 mM acetate, pH 5.5 0.01% Tween 20  0.9% benzyl alcohol136 mM NaCl 0.25% phenol 0.45% phenol 0.25% m-cresol 0.18% methylparaben0.02% propylparaben  20 mM acetate, pH 5.5 0.01% F68  0.9% benzylalcohol 136 mM NaCl 0.25% phenol 0.45% phenol 0.25% m-cresol 0.18%methylparaben 0.02% propylparaben

[0087] Experimental Design

[0088] All rhNGF multi-dose liquid formulations prepared were sterilefiltered through 0.22 μm filter prior to filling. Each formulations wereaseptically filled into Type 1, clear glass, 3 cc Wheaton vials with afill volume of 1.8 mL. Vials were stoppered with 13 mm Purcoat stoppersand hand crimped with 13 mm aluminum flip-off caps.

[0089] For the preservative screening study, samples were stored at roomtemperature for 24 hours to determine physical compatibility. For theformulation development study, samples were stored at −70, 5, 25 and 40°C. At each time point, one sample/formulation/temperature was assayed.

[0090] Agitation studies were carried out at room temperature on thecurrent 2 mg/mL rhNGF formulation, the multi-dose formulations thatcontain either 0.9% benzyl alcohol or 0.25% phenol in the absence ofsurfactant, and the 0.1 mg/mL rhNGF control that contains no 24.surfactant and preservative. A 3 cc vial of each formulation tested wassecured to a laboratory bench top shaker (Glas-Col) and agitated at 80rpm for 6 and 24 hours. Samples collected after 6 and 24 hours ofshaking were assayed by SE-HPLC, RP-HPLC, ELISA and RRA.

[0091] Freezing and thawing cycling was performed on the sameformulations that used for agitation studies. One vial from eachformulation tested was placed in −70° C. freezer and allowed to freezefor 24 hours. After 24 hours of freezing, samples were thawed at 5° C.for 24 hours. This freezing and thawing procedure was repeated up to 3times. Samples collected at the end of the third cycle were assayed bySE-HPLC, RP-HPLC, ELISA and RRA.

[0092] The effect of light on stability of rhNGF was studied on the sameformulations that used for agitation studies. One vial from eachformulation was placed in a light box (Form a Scientific, Model 3890)under high intensity fluorescent light for 5 weeks. Control vialswrapped with aluminum foil were also placed in the light box. Lightintensity was 20,000 lux which was about 15-20 times that of indoorfluorescent light, and the temperature of the light box was maintainedat 28° C. Samples were assayed at 2 and 5 weeks by SEC-HPLC, ELISA andRRA.

[0093] Analytical Methodology

[0094] A. UV Analysis. rhNGF concentration was determined by scanningfrom 240 to 360 nm using an HP 8452A UV-Vis spectrophotometer.Formulation buffer was used as a reference to blank the instrument, andthe protein concentration in mg/mL was calculated from (A280-320)/1.5,where 1.5 is the extinction coefficient of rhNGF in mL/(mg·cm).

[0095] B. HPLC Analysis. The following HPLC methods were used.Reversed-Phase HPLC column: YMC C4, 5 μm, 4.6 × 250 mm mobile phase: A:0.05% (v/v) TFA, water B: 0.05% (v/v) TFA, 100% AcCN gradient: 25-27% B(26′), 27-50% B (4′), 50-80% B (1′), 80-25% B (4′), 25% B (20′) flowrate: 1 mL/min run time: 55 min temp: 25° C. LC: HP-1090 detection: 214,280 nm injection: 15 μg

[0096] Size Exclusion HPLC column: Tosohaas TSK 2000SWXL, 5 μm, 7.8 ×300 mm mobile phase: 0.2 M potassium phosphate, 0.45 M KCl, pH 7.0gradient: isocratic flow rate: 1.0 mL/min run time: 30 min temp: ambientLC: HP-1090 detection 214, 280 nm injection: 15 μg

[0097] Cation Exchange HPLC column: Tosohaas TSK SP-5PW, 10 μm, 7.5 × 75mm mobile phase: A: 10 mM sodium phosphate, 10% (v/v) AcCN, pH 7.0 B:A + 1 M ammonium chloride gradient: 10-40% B (60′), 40-60% B (5′),60-10% B (1′), 71-86% B (15′) flow rate: 0.5 mL/min run time: 86 mintemp: 35° C. LC: HP-1090 detection 214 nm injection: 15 μg

[0098] C. ELISA. This assay with a range of 0.39-6.25 ng/mL was carriedout by Immunoassay Services (Test Procedure Code SNGF:1 of Genentech,Inc.). Each rhNGF sample was diluted in assay diluent to two targetconcentrations of 5 and 2.5 ng/mL, and each dilution was submitted inmicronic tubes in triplicate. The protein concentration in mg/mL wasnormalized to a −70° C. internal reference standard which was submittedfor the same assay.

[0099] D. Radioreceptor Assay (RRA). This assay measures the ability ofunlabeled rhNGF to compete with ¹²⁵I-rhNGF for receptor binding on PC-12cells. This assay was carried out by Bioassay Service (Genentech, Inc.Test Procedure SNGF:6) and has a range of 3-80 ng/mL. Each rhNGF samplewas diluted in assay diluent to two target concentrators of 25 and 12.5ng/mL, and each dilution was submitted in micronic tubes in duplicate.The protein concentration in mg/mL was normalized to a −70° C. internalreference standard which was submitted for the same assay.

[0100] E. PC-12 Cell Survival Bioassay. This assay determines theability of rhNGF to bind to its receptors and generate intracellularsignals that result in the survival of PC-12 cells under serum-freeculture conditions. This assay was carried out by Bioassay Service (TestProcedure SNGF:7) and has a range of 0.24-30 ng/mL. The active proteinconcentration in mg/mL was normalized to a −70° C. internal referencestandard which was submitted for the same assay.

[0101] F. Visual Inspection. Visual inspection was performed on allformulations in vials at the time of sampling. Samples were observed forsolution clarity, color, opalescence and particulate formation.

[0102] G. pH Determination. pH of all formulations was determined ateach timepoint using a radiometer (model PHM82, Radiometer America Inc.)and a micro-electrode (model M1-410, Microelectrodes, Inc.). Standardsolutions of pH 4.01 and pH 7.00 were used for the standardization andcalibration of the radiometer prior to pH measurement.

[0103] H. Preservative Effectiveness Test. The lead rhNGF multi-doseliquid formulations which were stable at 5° C. for 6 months were sent toNorthview Lab for bacterial challenge testing based on USP and EPstandard criteria.

[0104] I. Circular Dichroism (CD) Analysis. An A VIV® spectropolarimeterModel 60 DS equipped with water bath and data processor was used tomeasure circular dichroism. Measurements were made at 20° C. Quartzcuvettes of 1.0 cm cell path length was used for measuring near-UV CD.The CD spectra was taken at 0.2 nm intervals, with a 0.5 nm bandwidth,and 3.0 second averaging time. Each sample for CD measurement was takencontinuously for 24 hours. The CD data were expressed as the meanresidue ellipticity [q], degree·cm2/decimole, using the mean residueweight of 120 for rhNGF.

[0105] Results

[0106] A preservative screening study was first performed to examine thephysical compatibility of several commonly used preservatives with rhNGFat 0.1 mg/mL in the 20 mM sodium acetate formulation at pH 5.5. Thesepreservatives include benzyl alcohol, phenol, m-cresol, methylparabenand propylparaben. In addition, the physical compatibility of thesepreservatives with rhNGF in the acetate formulation with the presence ofsurfactants such as polysorbate 20 and pluronic acid (F68) was alsostudied. The physical compatibility results are shown in Table 4. TABLE4 List of rhNGF Liquid Formulations Selected for Long Term StabilityTesting I. Current liquid formulation  1. 2 mg/mL rhNGF in 10 mMacetate, 142 mM sodium chloride, pH 5.5 II. Control liquid formulations(no preservative)  1. 0.1 mg/mL rhNGF in 20 mM acetate, 136 mM sodiumchloride, pH 5.5  2. 0.1 mg/mL rhNGF in 20 mM acetate, 136 mM sodiumchloride, 0.01% F68, pH 5.5 III. Multi-dose liquid formulations  1. 0.1mg/mL rhNGF in 20 mM acetate, 136 mM sodium chloride, 0.9% benzyl   alcohol, pH 5.5  2. 0.1 mg/mL rhNGF in 20 mM acetate, 136 mM sodiumchloride, 0.25%    phenol, pH 5.5  3. 0.1 mg/mL rhNGF in 20 mM acetate,136 mM sodium chloride, 0.01% F68,    0.9% benzyl alcohol, pH 5.5  4.0.1 mg/mL rhNGF in 20 mM acetate, 136 mM sodium chloride, 0.01% F68,   0.25% phenol, pH 5.5

[0107] Among the preservatives used for screening, they are allphysically compatible with rhNGF at 0.1 mg/mL in the acetate formulationat pH 5.5. In the presence of polysorbate 20 at 0.01% in the sameformulation, only benzyl alcohol and phenol at final concentrations of0.9% and 0.25% respectively were physically compatible with rhNGF.Phenol at 0.45% and m-cresol at 0.25% each formed a cloudy solution withrhNGF in the acetate formulation in the presence of polysorbate 20. TherhNGF solution also became slightly opalescent upon the addition ofmethylparaben at 0.18% or propylparaben at 0.02% to the polysorbate 20containing acetate formulation. On the other hand, pluronic acid at0.01% in the same formulation did not cause any physical incompatibilitybetween rhNGF and all the preservatives tested.

[0108] Based on the preservative screening study results, several rhNGF,multi-dose liquid formulations containing either 0.9% benzyl alcohol or0.25% phenol in 20 mM acetate at pH 5.5 with and without 0.01% F68 wereset up for long term stability study. A list of these formulations weregiven in Table 5. TABLE 5 Physical Compatibility of Preservatives with0.1 mg/mL rhNGF Liquid Formulations Formulation buffer SurfactantPreservative Results  20 mM acetate, none  0.9% benzyl alcohol co/cl pH5.5 0.25% phenol co/cl 136 mM NaCl 0.45% phenol co/cl 0.25% m-cresolco/cl 0.18% methylparaben co/cl 0.02% propylparaben co/cl  20 mMacetate, Tween 20  0.9% benzyl alcohol co/cl pH 5.5 0.01% 0.25% phenolco/cl 136 mM NaCl 0.45% phenol cloudy 0.25% m-cresol cloudy 0.18%methylparaben sl. opal 0.02% propylparaben sl. opal  20 mM acetate,0.01% F68  0.9% benzyl alcohol co/cl pH 5.5 0.25% phenol co/cl 136 mMNaCl 0.45% phenol co/cl 136 mM NaCl 0.25% m-cresol co/cl 0.18%methylparaben co/cl 0.02% propylparaben co/cl

[0109] Stability of rhNGF in these formulations was assayed by thefollowing techniques: SE-HPLC, RP-HPLC, IE-HPLC, ELISA, radioreceptorassay (RRA), PC-12 cell survival bioassay, pH, and visual inspection.The acceptability of a multi-dose liquid formulation for rhNGF will bebased on comparison to the current liquid formulation which consists of2 mg/mL rhNGF in 10 mM sodium acetate at pH 5.5, and 142 mM sodiumchloride. In the other word, the preserved formulation should be asstable as the current liquid formulation. Results obtained to daterepresent 12 months at −70 and 5° C., 9 months at 25° C., and 3 monthsat 40° C. stability monitoring data.

[0110] Size-Exclusion Chromatography. Size-exclusion HPLC was employedto detect and quantitate aggregate formation in the rhNGF multi-doseliquid formulations as well as their control formulations which containno preservative. Using this technique, rhNGF elutes as dimer (main peak)at a retention time of 8.6 minutes. Benzyl alcohol and phenol elute at16 and 19 minutes respectively. The appearance of leading shoulder onthe dimer main peak indicates the presence of aggregate of highermolecular weight. The data in Table 6 shows that rhNGF is stable toaggregate formation in all formulations containing 0.9% benzyl alcoholas preservative. TABLE 6 Effect of preservative on aggregation of 0.1mg/mL rhNGF in liquid formulations was determined by SEC-HPLC. Sampleswere stored at 5° C. for 12 months, 25° C. for 9 months and 40° C. for 3months. % Aggregate Formulation buffer Surfactant Preservative 5° C. 25°C. 40° C.  10 mM acetate, pH 5.5 none none 0 0.2 0.4 145 mM NaCl, 2mg/mL  20 mM acetate, pH 5.5 none none 0 0 0 136 mM NaCl, 0.1 mg/mL  20mM acetate, pH 5.5 none  0.9% benzyl. 0 0 0 136 mM NaCl, 0.1 mg/mL alc. 20 mM acetate, pH 5.5 none 0.25% phenol 0 0.4 0.5 136 mM NaCl, 0.1mg/mL  20 mM acetate, pH 5.5 0.01% F68 none 0 0 0 136 mM NaCl, 0.1 mg/mL 20 mM acetate, pH 5.5 0.01% F68  0.9% benzyl 0 0 0 136 mM NaCl, 0.1mg/mL alc.  20 mM acetate, pH 5.5 0.01% F68 0.25% phenol 0 0.5 0.5 136mM NaCl, 0.1 mg/mL

[0111] A small amount of aggregate (less than 1%) was detected in thephenol containing formulations (with and without 0.01% F68 assurfactant) after 3 months at 40° C. and 9 months at 5° C. Total proteinrecovery of these samples, compared to their −70° C. controls, was givenin Table 7. TABLE 7 Quantitation of total rhNGF by SE-HPLC. Samples werestored at 5° C. for 12 months, 25° C. for 9 months and 40° C. for 3months. % Recovery Formulation buffer Surfactant Preservative 5° C. 25°C. 40° C.  10 mM acetate, pH 5.5 none none 102 102 102 145 mM NaCl, 2mg/mL  20 mM acetate, pH 5.5 none none 101 101 101 136 mM NaCl, 0.1mg/mL  20 mM acetate, pH 5.5 none  0.9% benzyl alc. 102 99 101 136 mMNaCl, 0.1 mg/mL  20 mM acetate, pH 5.5 none 0.25% phenol 99 97 98 136 mMNaCl, 0.1 mg/mL  20 mM acetate, pH 5.5 0.01% F68 none 101 101 98 136 mMNaCl, 0.1 mg/mL  20 mM acetate, pH 5.5 0.01% F68  0.9% benzyl alc. 10199 99 136 mM NaCl, 0.1 mg/mL  20 mM acetate, pH 5.5 0.01% F68 0.25%phenol 100 97 97 136 mM NaCl, 0.1 mg/mL

[0112] Current formulation, controls and benzyl alcohol containingformulations had 99% or greater protein recovery after 9 months at 25°C., while phenol containing formulations had 97% for the same storagetime and temperature. These results indicate that rhNGF is morecompatible and stable with benzyl alcohol than phenol in allformulations studied.

[0113] Reversed-Phase HPLC. The rhNGF used in this study consists ofmainly 118/118 homodimer and a small amount of 120/120 homodimer. Underthe conditions of reversed-phase chromatography, the two rhNGF dimericforms are dissociated and their monomers are separated. RP-HPLCseparates the rhNGF monomers based on the hydrophobicity of eachspecies. The 118 monomer which is more hydrophobic than the 120 monomerelutes at a retention time of 23 minutes. The 120 monomer elutes as asmall peak in front of the 118 monomer peak. Comparison of RP-HPLCchromatograms of rhNGF in the benzyl alcohol preserved formulationcontaining no surfactant at 5, 25, and 40° C. are shown in FIG. 8. Thedegradation of rhNGF stored at elevated temperatures was mainly due tothe formation of iso-aspartate, loss in 118 and 120 monomer peak areas,clip formation and increase in misfolded rhNGF as determined by RP-HPLC.The mono- and di-oxidized rhNGF peaks and the deamidated rhNGF peakremain unchanged. In this study, rhNGF is defined as the sum of the 118and 120 monomer peak areas by RP-HPLC, and the results are reported aspercent rhNGF remaining as compared to the −70° C. controls.

[0114] Decrease in percent protein remaining due to the loss of 118 and120 monomer peak areas assayed by RP-HPLC is the major degradation forrhNGF in liquid formulation. At 5° C., the stability of rhNGF inmulti-dose formulations as determined by RP-HPLC are essentiallyequivalent to the non-preserved control formulations as well as thecurrent formulation (more than 95% rhNGF remaining after 12 months)except for the phenol preserved formulation containing 0.01% F68 (FIG.9). This formulation had slightly less percent rhNGF remaining (93%)after 12 months at 5° C. At 25° C., rhNGF is obviously less stable inthe presence of 0.25% phenol than 0.9% benzyl alcohol as preservative inthe 20 mM acetate formulation at pH 5.5 (FIG. 10). The combination ofphenol and F68 in the acetate formulation caused more degradation of theprotein than the presence of phenol alone.

[0115] Iso-aspartate formation of rhNGF in liquid form is time andtemperature dependent. The rate of iso-aspartate formation increaseswith increase in time and temperature. At 5° C., all formulations show asimilar rate of iso-aspartate formation (FIG. 11). There was about 1.5%iso-aspartate formed in all rhNGF multi-dose formulations and theirnon-preserved control formulations after 12 months at 5° C. However, therate of iso-aspartate formation is slightly higher in the rhNGFformulations preserved with 0.9% benzyl alcohol than the controlformulations and phenol preserved formulations stored at 25° C. (FIG.12). Since iso-aspartate formation of rhNGF does not affect thebioactivity of the protein, the effect of preservative on iso-aspartateformation of rhNGF is not a major concern.

[0116] Cation Exchange Chromatography. IE-HPLC chromatograms for rhNGFin the current formulation at 3 months at 5, 25, and 40° C. are shown inFIG. 13. There are three major peaks observed. The predominant peak isthe 118/118 dimer (peak b) which elutes at about 48 minutes. The peak cbehind the main peak is from a serine to glycine substitution atposition 1 in one of the two dimer chain. The peak a in front of themain peak is believed to be the oxidized 118/118 and oxidizedN-terminally clipped rhNGF. At elevated temperatures (25 and 40° C.),degradation of rhNGF as determined by IE-HPLC is characterized by thedecrease in peak areas of the 118/118 main peak and the serine toglycine substituted 118/118 dimer and the increase in peak a area. Inthis study, rhNGF is defined as the sum of the 118/118 dimer (peak b)and one chain serine to glycine dimer (peak c) peak areas by IE-HPLC,and the results are reported as percent rhNGF remaining as compared tothe −70° C. controls.

[0117]FIGS. 14 and 15 show the percent rhNGF remaining in all rhNGFformulations by IE-HPLC after 12 months at 5° C. and 9 months at 25° C.,respectively. At 5° C., the peak area of peaks b and c for all rhNGFformulations remained unchange after 12 months. At 25° C., all rhNGFformulations show a similar rate of degradation, and there was nosignificant difference in stability between the multi-dose formulationsand the control formulations as assessed by IE-HPLC.

[0118] ELISA. The data in Table 8 show the percent rhNGF remaining at 5,25 and 40° C. after 12, 9 and 3 months of storage, respectively. TABLE 8Stability of current and selected multi-dose liquid formulations forrhNGF determined by ELISA after 12 months at 5° C., 9 months at 25° C.,and 3 months at 40° C. % rhNGF ^(a)Remaining Formulation bufferSurfactant Preservative 5° C. 25° C. 40° C.  10 mM acetate, pH 5.5 nonenone 101.2 89.1 102.2 145 mM NaCl, 2 mg/mL  20 mM acetate, pH 5.5 nonenone 97.8 102.0 94.4 136 mM NaCl, 0.1 mg/mL  20 mM acetate, pH 5.5 none 0.9% benzyl 103.1 92.9 97.1 136 mM NaCl, 0.1 mg/mL alc.  20 mM acetate,pH 5.5 none 0.25% phenol 111.3 88.5 91.6 136 mM NaCl, 0.1 mg/mL  20 mMacetate, pH 5.5 0.01% F68 none 98.5 102.7 92.7 136 mM NaCl, 0.1 mg/mL 20 mM acetate, pH 5.5 0.01% F68  0.9% benzyl 101.9 92.6 87.7 136 mMNaCl, 0.1 mg/mL alc.  20 mM acetate, pH 5.5 0.01% F68 0.25% phenol 103.492.5 82.6 136 mM NaCl, 0.1 mg/mL

[0119] Results were normalized to the −70° C. controls stored at thesame temperature for the same period of time. There were no significantdifference between the benzyl alcohol and phenol preserved formulationeither in the presence or absence of 0.01% F68 as surfactant for alltemperatures and time points studied.

[0120] Radioreceptor Binding Activity (RRA). The RRA results arepresented in Table 9 and are normalized to the −70° C. controls. TABLE 9Stability of current and selected multi-dose liquid formulations forrhNGF determined by RRA after 12 months at 5° C., 9 months at 25° C.,and 3 months at 40° C. % rhNGF ^(a)Remaining Formulation bufferSurfactant Preservative 5° C. 25° C. 40° C.  10 mM acetate, pH 5.5 nonenone 111.3 121.5 74.9 145 mM NaCl, 2 mg/mL  20 mM acetate, pH 5.5 nonenone 100.6 106.5 82.1 136 mM NaCl, 0.1 mg/mL  20 mM acetate, pH 5.5 none 0.9% benzyl alc. 94.2 91.3 81.6 136 mM NaCl, 0.1 mg/mL  20 mM acetate,pH 5.5 none 0.25% phenol 82.0 72.5 68.8 136 mM NaCl, 0.1 mg/mL  20 mMacetate, pH 5.5 0.01% F68 none 92.9 79.2 80.8 136 mM NaCl, 0.1 mg/mL  20mM acetate, pH 5.5 0.01% F68  0.9% benzyl alc. 92.0 80.7 83.2 136 mMNaCl, 0.1 mg/mL  20 mM acetate, pH 5.5 0.01% F68 0.25% phenol 98.0 83.773.7 136 mM NaCl, 0.1 mg/mL

[0121] In the absence of 0.01% F68 in the acetate formulation at pH 5.5,the phenol preserved formulation had less percent protein remaining thanboth the benzyl alcohol preserved formulation and the controlformulation for all temperatures studied. In the presence of 0.01% F68in the acetate formulation at pH 5.5, rhNGF in the preserved (benzylalcohol or phenol) and the control formulation had lost about 20% of itsbioactivity at. 25 and 40° C. after 9 and 3 months, respectively. Theseresults suggest that phenol and F68 can affect the ability of rhNGF tobind to the NGF receptor on PC-12 cells. Therefore, benzyl alcohol at0.9% is a better choice of preservative for rhNGF in the acetateformulation containing no surfactant for multi-use purpose.

[0122] PC-12 Cell Survival Bioassay. In contrast to the RRA results, thePC-12 cell survival bioassay data in Table 10 show that there was nosignificant difference in potency of rhNGF in all formulations stored at5° C. for 12 months and 25° C. for 9 months. TABLE 10 Stability ofcurrent and selected multi-dose liquid formulations for rhNGF determinedby bioassay after 12 months at 5° C. and 9 months at 25° C. % rhNGF^(a)Remaining Formulation buffer Surfactant Preservative 5° C. 25° C. 10 mM acetate, pH 5.5 none none 101.7 96.1 145 mM NaCl, 2 mg/mL  20 mMacetate, pH 5.5 none none 84.3 113.7 136 mM NaCl, 0.1 mg/mL  20 mMacetate, pH 5.5 none  0.9% benzyl alc 102.2 97.3 136 mM NaCl, 0.1 mg/mL 20 mM acetate, pH 5.5 none 0.25% phenol 95.3 102.1 136 mM NaCl, 0.1mg/mL  20 mM acetate, pH 5.5 0.01% F68 none 101.3 95.9 136 mM NaCl, 0.1mg/mL  20 mM acetate, pH 5.5 0.01% F68  0.9% benzyl alc. 96.6 94.2 136mM NaCl, 0.1 mg/mL  20 mM acetate, pH 5.5 0.01% F68 0.25% phenol 97.896.4 136 mM NaCl, 0.1 mg/mL

[0123] The protein was found to be fully active in all formulations asdetermined by this bioassay. Therefore, the radioreceptor binding assayis a more stability indicating assay than the cell survival bioassay indetermining the bioactivity of rhNGF.

[0124] Solutions of all rhNGF formulations were clear and colorless tothe naked eyes (Table 11). Particulates were not observed in any of theformulations at all temperatures and timepoints. TABLE 11 pH and visualclarity of rhNGF formulations after 12 months at 5° C. and 9 months at25° C. Visual Formulation pH Clarity pH Visual Clarity buffer 5° C. 5°C. 25° C. 25° C.  10 mM acetate, pH 5.5 5.50 co/cl 5.40 co/cl 145 mMNaCl, 2 mg/mL  20 mM acetate, pH 5.5 5.54 co/cl 5.41 co/cl 136 mM NaCl,0.1 mg/mL  20 mM acetate, pH 5.5 5.52 co/cl 5.58 co/cl 136 mM NaCl, 0.1mg/mL  0.9% benzyl alc.  20 mM acetate, pH 5.5 5.49 co/cl 5.60 co/cl 136mM NaCl, 0.1 mg/mL 0.25% phenol  20 mM acetate, pH 5.5 5.47 co/cl 5.53co/cl 136 mM NaCl, 0.1 mg/mL 0.01% F68  20 mM acetate, pH 5.5 5.42 co/cl5.42 co/cl 136 mM NaCl, 0.1 mg/mL 0.01% F68, 0.9% benzyl alc.  20 mMacetate, pH 5.5 5.48 co/cl 5.41 co/cl 136 mM NaCl, 0.1 mg/mL 0.01% F68,0.25% phenol

[0125] pH Results. rhNGF formulated in 10 mM acetate, 142 mM sodiumchloride at either pH 5.0 or pH 5.8 had an increase in pH by 0.2 unitsduring the stability study. The multi-dose formulations and theircontrol formulations used in this study were formulated in 20 mM acetateat pH 5.5 which should provide a higher buffer capacity to prevent pHchange. Table 11 shows that pH remained unchange for all formulationsstudied.

[0126] Preservative Effectiveness Test. After 6 months of stabilitystudy, the most stable multi-dose formulation for rhNGF which consistsof 0.1 mg/mL rhNGF in 20 mM acetate at pH 5.5, 136 mM sodium chloride,and 0.9% benzyl alcohol was submitted for preservative efficacy testing.This lead formulation passed both the USP and EP (criteria A and B)after 6 months storage at 5° C.

[0127] Circular Dichroism (CD) Analysis. The presence of 0.9% benzylalcohol in various liquid interferon-gamma (rhIFN-g) formulationsinduces loss in circular dichroism signals in the near-UV region. Thenear-UV CD signal of rhIFN-g disappeared within 24 hours, indicatingthat there was a change in tertiary structure of the protein due to thepresence of benzyl alcohol. However, this phenomenon was not observed inthe rhNGF formulation preserved with 0.9% benzyl alcohol. After 24 hoursof the addition of the preservative, the near-UV CD spectrum remainedunchange, suggesting that there is no interaction between rhNGF andbenzyl alcohol in the acetate formation at pH 5.5. FIG. 16 shows thenear-UV CD spectrum of rhNGF, and FIG. 17 compares the rear-UV CDspectra of rhNGF in the presence and absence of benzyl alcohol after 24hours at 25° C. Due to the interference of benzyl alcohol at wavelengthbelow 275 nm, CD spectrum of rhNGF was scanned from 325 nm to 275 nmwhen the sample contained the preservative.

[0128] Stresses Testing Stability

[0129] 1. Agitation Studies. Shaker studies were performed to determinewhether it is necessary to add surfactant (F68) in the rhNGF multi-doseformulations at low protein concentration such as 0.1 mg/mL in order toprevent protein aggregation and maintain visual clarity of the solutionsduring agitation. The Data of Table 12 show that rhNGF at 0.1 mg/mL inthe 20 mM acetate formulation at pH 5.5 (with or without preservative)is quite stable to mechanical disruption such as shaking. This suggeststhat surfactant is not required in formulating rhNGF at 0.1 mg/mL asmulti-dose liquid form for stability purpose. TABLE 12 Effect ofagitation on stability of rhNGF multi-dose liquid formulations. Sampleswere shaken at 80 rpm for 6 and 24 hours at room temperature. % Monomer% Iso-Asp % NGF ELISA RRA Formulation Hours (SEC) (RP-HPLC) (RP-HPLC)(mg/mL) (mg/mL) 1 6 0 0.6 101.6 0.1 0.11 24 0.4 0.5 101.7 0.09 0.11 2 60 0.8 103.6 0.09 0.11 24 0 0.7 100.8 0.09 0.10 3 6 0 0.6 101.3 0.09 0.1024 0 0.6 101.0 0.09 0.10 4 6 0 0.6 100.8 0.09 0.10 24 0 0.7 101.0 0.090.10

[0130] 2. Freezing-Thawing Studies. Results on the effect of freezingand thawing on stability of 0.1 mg/mL rhNGF multi-dose liquidformulations are presented in Table 13. TABLE 13 Effect of freeze-thawon stability of rhNGF multi-dose liquid formulations. Freeze −70° C. %Aggregate % Iso-Asp % NGF ELISA RRA Formulation Thaw 5° C. (SEC)(RP-HPLC) (RP-HPLC) (mg/mL) (mg/mL) 1 3 cycles 0 0.9 102.1 0.09 0.10 2 3cycles 0 0.4 102.1 0.08 0.10 3 3 cycles 0 0.8 101.3 0.09 0.11 4 3 cycles0 0.5 101.8 0.09 0.10

[0131] After 3 cycles of freezing and thawing, the 0.1 mg/mL rhNGF inthe 20 mM acetate formulation at pH 5.5 as control and the twomulti-dose formulations containing either 0.9% benzyl alcohol or 0.25%phenol did not show any loss in stability of the protein. They are asstable as the current 2 mg/mL rhNGF liquid formulation after 3 cycles offreezing and thawing between −70 and 5° C.

[0132] 3. Light Compatibility Studies. Table 14 summarizes the effect oflight on stability of rhNGF in the current 2 mg/mL formulation, the 0.1mg/mL rhNGF control formulation, and the benzyl alcohol or phenolpreserved 0.1 mg/mL rhNGF formulations. TABLE 14 Effect of light onstability of rhNGF multi-dose liquid formulations. Samples wereilluminated at a light intensity of 20,000 lux at 28° C. Conc. Storage %Aggregate ELISA RRA Formulation (mg/mL) Condition Weeks (SEC) (mg/mL)(mg/mL)  10 mM acetate pH5.5 2 Dark 2 0 2.20 2.00 145 mM NaCl 5 0.3 2.202.00  20 mM acetate pH5.5 0.1 Dark 2 0 0.10 0.11 136 mM NaCl 5 0 0.090.10  20 mM acetate pH5.5 0.1 Dark 2 0 0.10 0.11 136 mM NaCl, 5 0 0.100.10  0.9% benzyl alcohol  20 mM acetate pH5.5 0.1 Dark 2 0 0.10 0.10136 mM NaCl, 5 0.2 0.10 0.10 0.25% phenol  10 mM acetate pH5.5 2 Light 20.4 2.20 2.40 145 mM NaCl 5 1.6 2.00 1.80  20 mM acetate pH5.5 0.1 Light2 0 0.10 0.10 136 mM NaCl 5 0.3 0.09 0.09  20 mM acetate pH5.5 0.1 Light2 0 0.10 0.10 136 mM NaCl, 5 0.2 0.09 0.09 0.9% benzyl alcohol  20 mMacetate pH5.5 0.1 Light 2 0.7 0.09 0.10 136 mM NaCl, 5 12.1 0.07 0.040.25% phenol

[0133] After storage for 2 weeks in the light box, there was nosignificant loss in stability of the protein in all formulationsstudied. However, after 5 weeks of storage in the light box, SE-HPLCindicated an increase in aggregate formation occurred in the currentformulation (1.6%). Aggregate formation was even more pronounced in thephenol preserved formulation (12.1%) after 5 weeks exposure to light.There was also a 30% loss in protein concentration and 60% inbioactivity in the light exposed phenol containing formulation asdetermined by ELISA and RRA, respectively. Both benzyl alcohol preservedformulation and the 0.1 mg/mL rhNGF control formulation were stableafter exposure to light for 5 weeks. All control vials wrapped withaluminum foil were stable after 5 weeks of storage in the light box.These results suggest that rhNGF is more sensitive to light at higherprotein concentration (2 mg/mL) than at lower protein concentration (0.1mg/mL) in the acetate formulation at pH 5.5. In the presence of phenol,rhNGF degrades more faster upon light exposure.

[0134] All 0.1 mg/mL rhNGF multi-dose liquid formation at pH 5.5 arestable at 5° C. for 12 months. At 25° C., the formulations (with orwithout F68) using 0.25% phenol as preservative were less stable thanthe formulations using 0.9% benzyl alcohol. 0.1 mg/mL rhNGF Formulationsat pH 5.5 containing surfactant (F68) are as stable as the formulationscontaining no surfactant.

[0135] The lead multi-dose formulation for rhNGF is 0.1 mg/mL protein in20 mM acetate, pH 5.5, 136 mM NaCl and 0.9% benzyl alcohol filled in 3cc vial with 1.8 mL filled. This formulation passed both the USP and EPpreservative efficacy testing after 6 month storage at 5° C.

[0136] rhNGF at 0.1 mg/mL formulated in 20 mM acetate, 136 mM NaCl pH5.5 is as stable as the current 2 mg/mL liquid formulation.

[0137] Agitation has no effect on stability of rhNGF, with regardless toprotein concentration or excipient in the formulation.

[0138] rhNGF is more stable in the dark than in the light especially ifthe formulation contains phenol as preservative.

[0139] rhNGF at 2 mg/mL in the current formulation and at 0.1 mg/mL inthe multi-dose liquid formulations can undergo at least 3 cycles offreezing (−70° C.) and thawing (5° C.) without any adverse effect on thestability of the protein.

CITED REFERENCES

[0140] 1. H. Thoenen and Y. A. Barde. Physiology of nerve growth factor.Physiol. Rev. 60:1284-1335 (1980).

[0141] 2. S. C. Apfel, R. B. Lipton, J. C. Arezzo, and J. A. Kessler.Nerve growth factor prevents toxic neuropathy in mice. Ann. Neurol.28:87-90 (1991)

[0142] 3. S. C. Apfel, J. C. Arezzo, L. A. Lipson, and J. A. Kessler.Nerve growth factor prevents experimental cisplatin neuropathy. Ann.Neurol. 31:76-80 (1992).

[0143] 4. B. G. Petty, D. R. Cornblath, B. T. Adornato, V. Chaudhry, C.Flexner, M. Wachsman, D. Sinicropi, L. E. Burton, S. J. Peroutka. Theeffect of systemically administered recombinant human nerve growthfactor in healthy human subjects. Ann. Neurol. 36:244-246 (1994).

[0144] 5. N. Q. McDonald, R. Lapatto, J. Murray-Rust, J. Gunning, A.Wlodawer, and T. L. Blundell. New protein fold revealed by a 2.3 Åresolution crystal structure of nerve growth factor. Nature 354:411-414(1991).

[0145] 6. M. A. Bothwell and E. M. Shooter. Dissociation equilibriumconstant of b nerve growth factor. J. Biol. Chem. 252:8532-8536 (1977).

[0146] 7. D. E. Timm, P. L. de Haseth, and K. E. Neet. Comparativeequilibrium denaturation of the neurotrophins: nerve growth factor,brain-derived neurotrophic factor, neurotrophin 3, and neurotrophin 4/5.Biochem. 33:4667-4676 (1994).

[0147] 8. C. H. Schmelzer, L. E. Burton, W.-P. Chan, E. Martin, C.Gorman, E. Canova-Davis, V. T. Ling, M. B. Sliwkowski, G. McCray, J. A.Briggs, T. H. Nguyen, and G. Polastri. Biochemical characterization ofrecombinant human nerve growth factor. J. Neurochem. 59:1675-1683(1992).

[0148] 9. J. B. Moore, and E. M. Shooter. The use of hybrid molecules ina study of the equilibrium between nerve growth factor monomers anddimers. Neurobiol. 5:369-381 (1975).

[0149] 10. L. A. Greene. A quantitative bioassay for nerve growth factoractivity employing a clonal pheochromocytoma cell line. Brain Res.133:350-353 (1977).

[0150] 11. K. Reed and S. Yalkowsky. Lysis of human red blood cells inthe presence of various cosolvents. III. The relationship betweenhemolytic potential and structure. J. Parenter. Sci. Technol. 41:37-39(1987)

[0151] 12. D. E. Timm and K. E. Neet. Equilibrium denaturation studiesof mouse b-nerve growth factor. Prot. Sci. 1:236-244 (1992).

[0152] 13. E. Canova-Davis, V. Ling, M. Eng, and S. Skieresz.Amino-terminal serine to glycine post-translational modificationobserved in nerve growth factor biosynthesized in Chinese hamster ovarycells. In Peptides: Chemistry, Structure and Biology, Escom SciencePublishers, Leiden, The Netherlands, pp. (1993). (Proceedings of theThirteenth American Peptide Symposium, Edmonton, Alberta, Canada, Jun.20-25, 1993)

[0153] 14. L. R. De Young, J. A. Briggs, and M. F. Powell Temperatureand pH dependence of recombinant human nerve growth factor dimerdissociation Biophys. J. 66: A401 (1994)

What is claimed is:
 1. A pharmaceutical composition, comprising apharmaceutically effective amount of nerve growth factor and apharmaceutically acceptable acetate-containing buffer.